Abstract
Molecular therapy using small interfering RNA (siRNA) shows great promise in the development of novel therapeutics for cancer. Although various approaches have been developed for in vivo delivery of siRNAs into tumors, stability of siRNA in blood circulation, and low efficiency of siRNA delivery into tumor cells are the major obstacles for further translation into cancer therapeutics. In this protocol, we describe methods of the production of shRNA expressing DNA nanocassettes by PCR amplification of double-stranded DNA fragments containing a U6 promoter and a shRNA gene. Those DNA nanocassettes can be conjugated to the polymer coating of nanoparticles that are targeted to cellular receptors highly expressed in tumor cells, such as urokinase plasminogen activator receptor (uPAR), for targeted delivery and receptor mediated internalization of shRNA expressing DNA nanocassettes. Methods for in vitro and in vivo evaluation of target specificity and gene-knockdown effect are also provided.
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Devi GR (2006) siRNA-based approaches in cancer therapy. Cancer Gene Ther 13(9):819–829
Kanasty R, Dorkin JR, Vegas A, Anderson D (2013) Delivery materials for siRNA therapeutics. Nat Mater 12(11):967–977
Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8(2):129–138
Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16(8):948–958
Cho YS, Lee GY, Sajja HK, Qian W, Cao Z, He W, Karna P, Chen X, Mao H, Wang AY, Yang L (2013) Targeted Delivery of siRNA-Generating DNA Nanocassettes Using Multifunctional Nanoparticles. Small 9(11):1964–1973
Gao X, Cui Y, Levenson RM, Chung LW, Nie S (2004) In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 22(8):969–976
Peng XH, Qian X, Mao H, Wang AY (2008) Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy. Int J Nanomedicine 3(3):311
Hermanson GT (2008) Bioconjugate techniques (second edition). London, p.219
Lee GY, Qian WP, Wang L, Wang YA, Staley CA, Satpathy M, Nie S, Mao H, Yang L (2013) Theranostic nanoparticles with controlled release of gemcitabine for targeted therapy and MRI of pancreatic cancer. ACS Nano 7(3):2078–2089
Yang L, Peng XH, Wang YA, Wang X, Cao Z, Ni C, Karna P, Zhang X, Wood WC, Gao X, Nie S, Mao H (2009) Receptor-targeted nanoparticles for in vivo imaging of breast cancer. Clin Cancer Res 15(14):4722–4732
Yang L, Mao H, Cao Z, Wang YA, Peng X, Wang X, Sajja HK, Wang L, Duan H, Ni C, Staley CA, Wood WC, Gao X, Nie S (2009) Molecular imaging of pancreatic cancer in an animal model using targeted multifunctional nanoparticles. Gastroenterology 136(5):1514–1525
Dohi T, Okada K, Xia F, Wilford CE, Samuel T, Welsh K, Marusawa H, Zou H, Armstrong R, Matsuzawa S, Salvesen GS, Reed JC, Altieri DC (2004) An IAP-IAP complex inhibits apoptosis. J Biol Chem 279(33):34087–34090
Chen Y, Zhu X, Zhang X, Liu B, Huang L (2010) Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther 18(9):1650–1656
Spitzer S, Eckstein F (1988) Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. Nucleic Acids Res 16(24):11691–11704
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This work is supported by NIH U01 CA 151810 (Yang) grant.
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Chen, W., Yang, L. (2016). Targeted Delivery with Imaging Assessment of siRNA Expressing Nanocassettes into Cancer. In: Medarova, Z. (eds) RNA Imaging. Methods in Molecular Biology, vol 1372. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3148-4_4
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DOI: https://doi.org/10.1007/978-1-4939-3148-4_4
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3147-7
Online ISBN: 978-1-4939-3148-4
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